Epidermal growth factor receptor (EGFR) is a 170 kilodalton (kDa) membrane-bound protein expressed on the surface of epithelial cells. EGFR is a member of the growth factor receptor family of protein tyrosine kinases, a class of cell cycle regulatory molecules. (W. J. Gullick et al., 1986, Cancer Res., 46:285-292). EGFR is activated when its ligand (either EFR or TGFα) binds to the extracellular domain, resulting in autophosphorylation of the receptor's intracellular tyrosine kinase domain (S. Cohen et al., 1980, J. Biol. Chem., 255:4834-4842; A. B. Schreiber et al., 1983, J. Biol. Chem., 258:846-853).
EGFR is the protein product of a growth promoting oncogene, erbB or ErbB1, that is but one member of a family, i.e., the ERBB family of protooncogenes, believed to play pivotal roles in the development and progression of many human cancers. The ERBB family of oncogenes encodes four, structurally-related transmembrane receptors, namely, EGFR, HER-2/neu (erbB2), HER-3 (erbB3) and HER-4 (erbB4). Clinically, ERBB oncogene amplification and/or receptor overexpression in tumors have been reported to correlate with disease recurrence and poor patient prognosis, as well as with responsiveness in therapy. (L. Harris et al., 1999, Int. J. Biol. Markers, 14:8-15; and J. Mendelsohn and J. Baselga, 2000, Oncogene, 19:6550-6565).
EGFR is composed of three principal domains, namely, the extracellular domain (ECD), which is glycosylated and contains the ligand-binding pocket with two cysteine-rich regions; a short transmembrane domain, and an intracellular domain that has intrinsic tyrosine kinase activity. The transmembrane region joins the ligand-binding domain to the intracellular domain. Amino acid and DNA sequence analysis, as well as studies of nonglycosylated forms of EGFR, indicate that the protein backbone of EGFR has a mass of 132 kDa, with 1186 amino acid residues (A. L. Ullrich et al., 1984, Nature, 307:418-425; J. Downward et al., 1984, Nature, 307:521-527; C. R. Carlin et al., 1986, Mol. Cell. Biol., 6:257-264; and F. L. V. Mayes and M. D. Waterfield, 1984, The EMBO J., 3:531-537).
The binding of EGF or TGFα to EGFR activates a signal transduction pathway and results in cell proliferation. The dimerization, conformational changes and internalization of EGFR molecules function to transmit intracellular signals leading to cell growth regulation (G. Carpenter and S. Cohen, 1979, Ann. Rev. Biochem., 48:193-216). Genetic alterations that affect the regulation of growth factor receptor function, or lead to overexpression of receptor and/or ligand, result in cell proliferation. In addition, EGFR has been determined to play a role in cell differentiation, enhancement of cell motility, protein secretion, neovascularization, invasion, metastasis and resistance of cancer cells to chemotherapeutic agents and radiation. (M.-J. Oh et al., 2000, Clin. Cancer Res., 6:4760-4763).
Cancer results from a series of genetic alterations which can include activation of oncogenes that promote cell growth and/or the loss of tumor suppressor gene function, which inhibits cell growth. While mutation or overexpression of oncogenes produces proteins that can stimulate uncontrolled cell growth, mutation or deletion of tumor suppressor genes results in the production of non-functional proteins that no longer control cell proliferation. (W. P. Carney and J. Williams, 2001, AdvanceLaboratory, Women's Health, pp.1-3).
A number of reports have indicated that the overexpression of EGFR occurs in many tumors, cancers, and malignancies; specifically, in breast, squamous cell, head and neck, glioma, lung, gastric, pancreatic, bladder, cervix, ovarian and prostate cancers. (M.-J. Oh et al., 2000, Ibid.; (W. J. Gullick et al., 1986, Cancer Res., 46:285-292; B. Ozanne et al., 1986, J. Pathol., 149:9-14; S. J. McKenzie, 1991, Biochim. et Biophys. Acta, 1072:193-214; C. Wright et al., 1992, Br. J. Cancer, 65:118-212; G. N. Fuller et al., 1992, Mutation Res., 276:299-306; J. G. M. Klijn et al., 1992, Endocrine Rev., 13:3-17; S. Nicolson et al., 1991, Oncology, 1:43-52; and C. Wright et al., 1991, Br. J. Cancer, 63:967-970). The exact role of EGFR in tumorigenesis remains to be determined; however, some studies of breast cancer patients have suggested that elevated EGFR in tumors may correlate with poor prognosis and disease progression parameters, such as invasion or lymph node metastasis (J.-H. Choi et al., 1997, 87th Ann. Meeting AACR, Washington, D.C., Apr. 20-24, 1996, pp. 1879-1883; J. G. M. Klijn et al., 1992, Endocrine Reviews, 13:3-17; S. Nicolson et al., 1991, Diagnostic Oncology; 1:43-52; C. Wright et al., 1991, British Journal Cancer, 63:967-970; J. R. C. Sainsbury et al., 1987, Lancet, 1:1398-1402; M. A. Rios et al., 1988, Anticancer Research, 8:173-176; A. Ullrich et al., 1990, Cell, 61:203-212; R. Nicolson et al., 1991, British Journal Cancer, 63:146-150).
Recent work has shown that tumors that overexpress EGFR may be amenable to treatment with a variety of therapies that target EGFR. Such treatments include small molecule inhibitors of the kinase activity of EGFR (R. G. Dullea et al., 2000, Proc. 91st Ann. Meeting of the American Association for Cancer Research (AACR), 41 (Abstract #2550):401; J. M. Nelson et al., 2000, Proc. 91st Ann. Meeting AACR, 41 (Abstract #2533):241; T. O'Reilly et al., 2000, Proc. 91st Ann. Meeting AACR, 41 (Abstract #3069):481; and H. C. Kelly et al., 2000, Proc. 91st Ann. Meeting AACR, 41 (Abstract #3896):612); antisense oligonucleotides (L. Witters et al., 1999, Breast Cancer Research and Treatment, 53:41-50); and immunotherapies that act directly on EGFR (X-D Yang et al., 2000, Proc. 91st Ann. Meeting AACR, 41 (Abstract #3380):530; X-D Yang et al., 1999, Cancer Research, 59:1236-1243; and L. Milas et al., 2000, Clinical Cancer Research, 6:701-708). Such therapies can be combined with traditional chemotherapy regimens in order to increase therapeutic efficacy in a variety of cancers (T. Ohmori et al., 2000, Proc. 91st Ann. Meeting AACR, 41 (Abstract #3072):48236 and A. Budillon et al., 2000, Proceedings of the 91st Ann. Meeting AACR, 41 (Abstract #4910):773).
In addition to EGFR, Human Epidermal Growth Factor Receptor-2 (also termed HER-2, HER-2/neu, neu, or c-erbB-2), another cell surface growth factor receptor of the ERBB family of receptor tyrosine kinases, has also been reported to be associated with uncontrolled cell proliferation and cancers. Like EGFR, HER-2/neu is a transmembrane tyrosine kinase receptor expressed on epithelial cells. The full-length HER-2/neu polypeptide has a molecular weight of 185 kDa (p185).
The ECDs of both EGFR and HER-2/neu have been shown to be released from the cell surface and have been found to circulate in normal people and in cancer patients. The ECD or shed ECD of HER-2/neu is a glycoprotein of between 97 and 115 kDa, referred to as p105 (W. P. Carney et al., 1991, J. Tumor Marker Oncol., 6(2):53-72). The primary ECD of EGFR is 110 kDa and is referred to as p110. Smaller circulating fragments of EGFR have also been reported. (A. J. Baron et al., 1999, Cancer Epidemiology, Biomarkers and Prevention, 8:129-137). patients. The shed ECD of HER-2/neu has been shown to be elevated in cancer patient serum (e.g., W. P. Carney et al., 1991, J. Tumor Marker Oncol., 6(2):53-72). Elevations in EGFR have mainly been documented at the tissue level, based on the analysis of the full length EGFR, p170. Increased EGFR ECD levels have been reported in the sera of cervical cancer patients. (M.-J. Oh et al., 2000, Clin. Cancer Res., 6:4760-4763).
Because both EGFR and HER-2/neu are expressed in at least 20-40%of women with breast, ovarian and cervical cancers, as well as in a wide variety of other cancers affecting both genders, it is a problem in the art to be able to accurately and sensitively identify, screen and monitor those individuals who are likely to respond, and who are responding to, or benefiting from, anti-EGFR therapy and/or anti-HER-2/neu therapy, conventional anti-cancer or anti-neoplastic treatments or therapies, or combination therapies, where applicable.
The present invention solves such a problem by providing a sensitive and reliable method, particularly an immunoassay method, to determine changed levels, particularly, decreased levels, of EGFR ECD in body fluid samples of cancer patients relative to those of normal individuals. Methods are also provided in which levels of EGFR ECD are determined in conjunction with the levels of other analytes, in particular, HER-2/neu, to diagnose and/or prognose disease progression and survival in cancer patients. In addition, the present invention is advantageous in that it is employed to monitor cancer patients undergoing cancer or anti-neoplastic treatments and therapies for cancers associated with overexpression of EGFR to assist in the determination of cancer treatment progress and patient outcome during the course of disease and/or anti-cancer therapy(ies).